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Biotechnology and Bioengineering

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Online ISSN: 1097-0290

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Print ISSN: 0006-3592

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Microvalve‐based 3D bioprinting device. (a) Schematic diagram of the structure. (b) Printer components. (c) Microvalve nozzle.
Microvalve Nozzle Working Principle and Simulation Diagram. (a) Microvalve Structure Diagram. (b) Microvalve Control Timing Diagram. (c) Nozzle Modeling Diagram. (d) Mesh Division Model.
Characterization of sodium alginate gels with different concentrations. (a) Shear‐thinning property. (b) Stress–strain plot. (c) Viscoelasticity: storage modulus (G′) and loss modulus (G″).
Simulation of different printing processes.
(a–c) Printing results of low viscosity materials in extrusion printing. (d–f) Printing results of low viscosity materials in microvalve printing. (g) On the first day, the microvalve printing scaffold was placed in PBS. (h) On the fourth day, the microvalve printing scaffold was placed in PBS, and there was no apparent change. (i) On the seventh day, the scaffold was intact when taken out of the PBS with tweezers.

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3D bioprinting technology and equipment based on microvalve control

September 2024

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358 Reads

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1 Citation

Rihui Kang

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Jiaxing Wu

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Rong Cheng

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[...]

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Shengbo Sang
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Aims and scope


Biotechnology and Bioengineering provides an international forum for biotech researchers. As the first biotechnology journal dedicated to the field, our publication has contributed significantly for over sixty years to the advancement of biochemical engineering science. Biotechnology and Bioengineering publishes Perspectives, Research Articles, Short Communications, and Reviews. Our journal publishes work which has both immediate and future impact on biotechnological processes.

Recent articles


Growth of bacteria in the different cultivation systems. The graph displays the glucose consumption (green squares, left) and cell dry weight (orange circles, right) of B. amyloliquefaciens, C. coralloides, S. griseochromogenes and S. cattleya during cultivation in SF, BSF, 24 DWP, 48 FP and 96 DWP. The bacteria were all cultivated in GYM medium for 5 days at 30°C. Differences in bioprocess parameters of the cultivation systems are presented in the boxes on the right. The bioprocess parameters were obtained from literature and manufacturer specifications. Samples were withdrawn each day in triplicates and displayed as means. We calculated a maximum standard deviation of 0.9 g/L for all cell dry weight measurements and 0.3 g/L for all glucose concentrations. Microscopic images of each bacteria were taken at the end of the cultivation from SF.
Impact of cultivation systems on metabolic footprint. A The bar chart displays the total number of observed MFs from B. amyloliquefaciens, C. coralloides, S. griseochromogenes and S. cattleya during cultivation in SF (red), BSF (black), 24 DWP (green), 48 FP (purple) and 96 DWP (orange). B The Venn diagrams analyze the occurrence of detected MFs. Untargeted metabolomics workflow was used to investigate MFs from the collected supernatant extracts of each sample. These MFs represent detected ions grouped with their observed retention time (rt_m/z). Only MFs with an abundance greater than 1000, available MS/MS fragmentation and detection in all triplicates were considered. MFs originating from the pre‐culture and medium control samples were removed.
Impact of cultivation systems on SM production profiles during cultivation. Hierarchical clustering analysis of all MFs formed during cultivation of B. amyloliquefaciens, C. coralloides, S. griseochromogenes and S. cattleya in SF, BSF, 24 DWP, 48 FP and 96 DWP. The resulting dendrogram displays the clustering of all observed MFs based on their production profiles over time. The heat map displays the production profiles of each MF and bacteria in the different cultivation systems (Figures S4–S6). The profiles are classified as early production (blue), production & degradation (red), late production (green) and no detection (grey). Each MF was labelled with a name, feature (rt_m/z) and annotation confidence level (Tier 1 – 4). In‐source fragments are marked with the name of the precursor ion and the feature. All m/z represents [M + H] +, unless specified otherwise.
Molecular Network of detected MFs from Streptomyces cattleya. The graph displays a molecular network, with the highest detected intensity of each node from S. cattleya in SF (red), BSF (black), 24 DWP (green), 48 FP (purple) and 96 DWP (orange), presented in a pie chart. The size of the pie chart displays the overall highest observed intensity in this study. The shape around the pie chart represents the production profiles early production (pentagon), production & degradation (square) and late production (diamond), resulting from the hierarchical clustering analysis of all detected MFs (Figure 3). The nodes represent MFs and are labeled with their chemical structure, name, feature (rt_m/z) and the annotation confidence level (Tier 1 – 4). In‐source fragments are marked with the name of the precursor ion and associated MFs. All MFs represent [M + H]+ unless specified otherwise.
Comparison of growth and SMs space of cultivation systems to STR. A The graph displays the glucose consumption (left) and cell dry weight (right) of S. griseochromogenes during cultivation in SF, BSF, 24 DWP, 48 FP and STR. S. griseochromogenes was cultivated in GYM medium for five days at 30°C. B The Venn diagram analyze the occurrence of detected MFs and the percentage of shared MFs in each cultivation systems to STR. C The molecular network displays the highest detected intensity of each node in SF (red), BSF (black), 24 DWP (green), 48 FP (purple) and STR (blue), presented as a pie chart. The shape around the pie chart represents the production profiles early production (pentagon), production & degradation (square), late production (diamond) and not detected (hexagon), which were derived from the hierarchical clustering analysis of all detected MFs in STR (Figure S12). The nodes represent MFs and are labelled with their chemical structure, name, the MFs (rt_m/z) and the annotation confidence level (Tier 1 – 4). In‐source fragments are indicated by the name of the precursor ion and the associated MFs. All MFs represent [M + H]+ unless otherwise specified.
Microtiter Plate Cultivation Systems Enable Chemically Diverse Metabolic Footprints During Bacterial Natural Product Discovery
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April 2025

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12 Reads

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Georg Hubmann

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Stephan Lütz

Rediscovery of known structures is a frequent problem in screening for bioactive bacterial natural products (NPs). Highly parallelized microtiter plate cultivation systems (MPCS) can improve the chance to discover novel NPs by testing a multitude of cultivation conditions simultaneously. An in‐depth analysis and comparison of cultivation systems for NP discovery, however, has not been carried out so far. We compared the growth and metabolic footprint of four distinct bacterial species in three MPCS, shake flasks, and stirred tank bioreactors (STR). While the big majority of the cultivation systems provided good growth, we found a considerable divergence in secondary metabolite (SM) formation. The SM space was approximated by the appearance of unique mass features (MFs) in the supernatant extracts throughout the cultivation period. Molecular network analysis was applied to visualize the changes from detected MFs at the molecular level. The cultivation systems had a minor impact on the unicellular growing Bacillus amyloliquefaciens. This impact was more pronounced for the tested filamentous bacteria, resulting in a diversified metabolic footprint. The maximal overlap of 31% of produced MFs indicates a lack of comparability between the cultivation systems, resulting in different entries of growth phases and the formation of associated SMs. The detected SMs and its derivatives exhibited structural modification depending on the cultivation system. A comparison of Streptomyces griseochromogenes NP profile revealed that MPCS yielded less divergent SM formation than shake flasks. Our comprehensive assessment is the first to demonstrate the impact of cultivation systems on the bacterial metabolic footprint, confirming that MPCS provide a robust platform for the parallelization of bacterial cultivations for the discovery of bacterial NPs and accessing the chemical NP space more broadly.


A Mathematical Model for Determining Probabilistic Design Space in Mesenchymal Stem Cell Passage Culture

April 2025

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2 Reads

With their many therapeutic functions, mesenchymal stem cells (MSCs) are promising sources for regenerative medicine. However, in the manufacture of MSCs, without a method for exploring the effects of long‐term passage on cell proliferation potentials, the design of passage culture processes is challenging. Here, for the process design of the MSC passage culture, we propose a model for predicting the growth rate as a function of the cumulative population doubling level (cPDL) for each passage. Three steps were implemented: (1) passage culture experiments to correlate apparent growth rate with cPDL were conducted, (2) a model for predicting the growth rate as a function of cPDL was developed, and (3) a model to design the passage culture of MSCs from bone marrow (BM‐MSCs) and umbilical cord (UC‐MSCs) with stochastic simulation was applied. Two design variables (passage number and harvesting time) were investigated to define feasible operation regions as probabilistic design spaces to meet three quality indicators (senescence level, confluency level, and total number of cells) with given probabilities. Consequently, 10 and 62 conditions out of 165 were identified as feasible for BM‐ and UC‐MSCs, respectively, which would contribute to the industrial MSC passage culture process design.


Modeling Analysis of Oxygen Transfer Efficiency in Rest Cell Catalysis for Extra High‐Titer Xylonic Acid Bioproduction

The conflict arising from high‐titer products and substantial oxygen requirements in aerobic bioconversion results in high‐viscosity and oxygen transfer bottlenecks in dynamically changing biosystems. Currently, in the bioproduction of xylonic acid (XA), strategies to address the oxygen transfer bottleneck predominantly focus on macro‐level modifications of the bioreactor. In this study, aiming at the high‐viscosity biosystem, the optimal rotational speed equation was established at the fluid level by quantitatively investigating the variations and limitations of fluid rheological characteristics, gas holdup, cell respiration rate, and volume transfer coefficient of broth under different concentrations and rotational speeds. Based on the cell respiration rate under the optimal rotation speed, the theoretical production performance was calculated, and 679.3 g/L XA was achieved with the productivity of 14.2 g/L/h by batch feeding mode. Verified using actual production under the same conditions as a control, 649.3 g/L XA was finally accumulated with a productivity of 13.5 g/L/h, which was equivalent to 95.8% of the theoretical production. The intensification strategy for oxygen transfer provided insightful ideas to overcome the stubborn obstacles of obligate aerobic catalysis. Moreover, the study offered technical assistance and application potential for the production of high‐titer XA from high‐viscosity sugar‐rich lignocellulosic hydrolysate.


Mechanical stretching of a cell‐sheet under the optical microscope. (A) Schematic illustration of the optical setup. The cell‐sheet was sustained by a pair of glass microneedles attached to both ends of the cell‐sheet above a glass‐bottom dish. Cells were simultaneously illuminated with blue (488 nm) and yellow‐green (561 nm) lasers through a spinning disc confocal unit. Green fluorescent protein (GFP) and red fluorescent protein (RFP) signals were reflected by a dichroic mirror and relayed to a dual‐view system to image both signals simultaneously with a single camera. DM, dichroic mirror; M, mirror; OBJ, objective lens. (B) Bright‐field images of a cell‐sheet during stretching. Glass microneedles were located at the left and the right (observed as black shadows in the first and the second images). Double‐sided arrow at the bottom indicates the stretching direction. Cell‐sheet was stretched to 200% of the slack length. Scale bar, 10 μm.
Remodeling of actin filaments observed in a cell‐sheet expressing red fluorescent protein (RFP)‐actin. (A) Sequential micrographs of fluorescence images of RFP‐actin during stretching. Note that the same cell can continuously be observed. (B–D) Enlarged images of (A) at t = 0 s (upper panels) and at t = 20 s (lower panels), illustrating the deformation of cell nucleus (B), the increase of dot‐like structures after stretching (C), and actin filaments newly appeared after stretching (D). Cells outlined in the same color or marked by colored stars in (A–D) indicate the same cells. Red arrowheads indicate actin filaments. Double‐sided arrows indicate the stretching direction. Scale bars in (A) and (B–D) are 20 and 10 μm, respectively. Independent experiments were performed in six cell‐sheets from different preparations, all of which gave similar results.
Formation of actin filaments and mechano‐sensing by α‐catenin in stretched cell‐sheets. (A) Alexa Fluor 546‐conjugated phalloidin was used to visualize actin filaments. Immunofluorescence staining of α‐catenin was achieved using a monoclonal antibody, α18, that recognizes α‐catenin in a force‐dependent manner (see “Stretched”). Images show the maximum intensity of the three optical slices from the bottom of the cell sheet. Scale bar, 100 μm. Double‐sided arrows indicate the stretching directions. Independent experiments were performed in two and four cell‐sheets from different preparations for non‐stretched and stretched conditions, respectively, all of which gave similar results. (B and C) Fluorescence micrographs of a cell‐sheet expressing green fluorescent protein (GFP)‐fused wild type α‐catenin (GFP‐wt‐α‐catenin) (B) or GFP‐fused deletion mutant of α‐catenin lacking the vinculin binding site (GFP‐697‐α‐catenin) (C), and RFP‐actin before and after stretching, held for 30 min. The overexpression of wt‐α‐catenin did not interfere with the formation of actin filaments upon stretching (C). Right panels are the magnified and merged views of the areas indicated by yellow rectangles to the left. Scale bars, 20 and 10 μm in left and right panels, respectively. Independent experiments were performed in eight cell‐sheets in (B) and four cell‐sheets in (C) from different preparations, all of which gave respectively similar results.
Assembly of adherens junction proteins observed in cell‐sheets. Two fluorescent proteins were simultaneously observed on a single camera. Double‐sided arrows indicate the stretching direction. (A) (Top) Sequential micrographs of a cell‐sheet expressing green fluorescent protein (GFP)‐β‐catenin and red fluorescent protein (RFP)‐actin before and after stretching, held for 10, 15, 20, and 30 min. (Bottom) Magnified views of the areas indicated by yellow rectangles in the top panels. Scale bars, 20 μm. (B) Fluorescence micrographs of a cell‐sheet expressing GFP‐β‐catenin and RFP‐actin before and after stretching, held for 30 min. For better S/N ratio, image was obtained only once before stretching and 30 min after stretching. The 17 cell‐sheets out of 19 independent experiments from different preparations gave similar results. (C) Western blot analysis using antibodies against phospho‐β‐catenin (S552), phospho‐β‐catenin (S675), phospho‐β‐catenin (S33/S37/T41), phospho‐β‐catenin (T41/S45), β‐catenin and α‐tubulin as a loading control. Extracts were obtained from cell‐sheets before and after induction of stretching and held for 1 min. Filled and open arrow heads indicate β‐catenin and nonspecific bands, respectively. (D and E) Fluorescence micrographs of a cell‐sheet expressing GFP‐vinculin and RFP‐actin (D) or RFP‐ β‐catenin (E) before and after stretching, held for 30 min. The five cell‐sheets out of eight and six cell‐sheets out of nine independent experiments from different preparations gave similar results in (D) and (E), respectively. In (B, D and E), right panels are magnified and merged views of the areas indicated by yellow rectangles to the left. Scale bars, 20 and 10 μm in left and right panels, respectively.
Assembly of focal adhesion proteins observed in cell‐sheets. (A and B) Fluorescence micrographs of a cell‐sheet expressing green fluorescent protein (GFP)‐p130Cas and red fluorescent protein (RFP)‐actin (A) or mCherry‐β‐catenin (B) before and after stretching, held for 30 min. The eight cell‐sheets out of 10 and 10 cell‐sheets out of 12 independent experiments from different preparations gave similar results in (A) and (B), respectively. (C) Western blot analysis using antibodies against phospho‐p130Cas (Y165), p130Cas, phospho‐FAK (Y397), FAK and α‐tubulin as a loading control. Extracts were obtained from cell‐sheets before and after stretching and held for 1 min. (D and E) Fluorescence micrographs of a cell‐sheet expressing GFP‐zyxin and RFP‐actin (D) or mCherry‐β‐catenin (E) before and after stretching, held for 30 min. Independent experiments were performed in 9 cell‐sheets in (D) and six cell‐sheets in (E) from different preparations, all of which gave respectively similar results. In (A, B, D and E), the right panels are the magnified and merged views of the areas indicated by yellow rectangles to the left. Scale bars, 20 and 10 μm in left and right panels, respectively.
Dynamic Remodeling of Mechano‐Sensing Complexes in Suspended Fibroblast Cell‐Sheets Under External Mechanical Stimulus

April 2025

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11 Reads

Freestanding cell‐sheets are valuable bio‐materials for use in regenerative medicine and tissue engineering. Because cell‐sheets experience various mechanical stimulations during handling, it is important to understand the responses of cells to these stimulations. Here, we demonstrate changes in the localization of various proteins during the stretching of fibroblast cell‐sheets. These proteins are known to be involved in mechano‐sensing. Upon stretching, actin filaments appear parallel to the stretching direction. At cell‐cell junctions, β‐catenin forms clusters that co‐localize with accumulated vinculin and zyxin as well as the actin filaments. The p130 Crk‐associated substrate, known to be present in focal adhesions, is also recruited to these clusters and phosphorylated. Our results suggest that mechano‐sensing machinery is formed at cell‐cell junctions when the cell‐sheets are stretched.


Combinatorial Metabolic Engineering for Enhanced Gibberellic Acid Biosynthesis in Fusarium fujikuroi

Gibberellic acid (GA3), a quintessential diterpenoid phytohormone, is indispensable in agronomic practices, horticulture, and the wine industry. This study implemented a combinatorial metabolic engineering strategy within Fusarium fujikuroi (F. fujikuroi) by integrating the potentiation of global regulatory factors (GRFs), and amplification of biosynthetic precursors, alongside dynamic modulation of cofactors with dissolved oxygen supply, to precisely enhance GA3 biosynthesis. Transcriptomic analyses revealed that positive GRFs (AreB, Hat1, and Ada3) enhanced carbon and nitrogen metabolism, increased biomass accumulation, and upregulated transcription levels of the GA3 biosynthetic gene cluster. The use of endogenous nitrogen‐responsive promoters ensured a balanced supply of cofactors and oxygen, thereby preventing the accumulation of terpenoid by‐products. These combinatorial metabolic engineering strategies presented in this study make a significant step toward the enhancement of GA3 yield (3.22 g/L) via submerged fermentation of F. fujikuroi, offering novel insights to enable high‐level biosynthesis of secondary metabolites in fungal chassis.


Optimized Biosynthetic Pathway for Nonnatural Amino Acids: An Efficient Approach for L‐2‐Aminobutyric Acid Production

L‐2‐Aminobutyric acid (L‐2‐ABA) is a nonnatural chiral α‐amino acid which is widely used in various chiral pharmaceuticals and medical intermediates. Currently, the microbial metabolic engineering approach to enable Escherichia coli to produce L‐2‐ABA autonomously exists the problem of low synthesis efficiency, limiting its large‐scale application. In this study, we successfully constructed a strain of E. coli that can produce L‐2‐ABA efficiently via multi‐pathway transformation. Firstly, the growth defect of the start strain was restored by the help of screening transcriptional regulators. To maximize the accumulation of L‐2‐ABA, enhancements were made to the main synthetic pathways as well as cofactor systems and energy supply. Subsequently, transport proteins associated with osmotic stress tolerance were modified to improve adaptability of the strain during fermentation. Ultimately, the titer of L‐2‐ABA reached 42.14 g/L through the final strain ABAT38 in a 5‐liter bioreactor, with a productivity of 0.40 g/L/h and a glucose conversion of 0.39 g/g, which exceeded the highest levels reported before. The strategies proposed in this study contribute to the production of L‐2‐ABA. At the same time, it has reference significance for the biosynthesis of related nonnatural amino acids with phosphoenolpyruvate as the intermediate metabolite.


Integrating Affinity Chromatography in the Platform Process for Adenovirus Purification

April 2025

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5 Reads

Adenoviral vectors (AdVs) are gaining prominence in cancer therapy and vaccine development, posing the need for a modern AdV manufacturing platform. Current AdV purification by ion‐exchange chromatography indeed struggles to achieve the product's yield and purity of processes that employ affinity technologies. Addressing these challenges, this study presents the first affinity‐based process that delivers high product yield and clearance of host cell proteins and DNA (HCPs and hcDNA) in two chromatography steps. The affinity capture utilizes resins functionalized with peptide ligands that target AdV hexon proteins (AEFFIWNA and TNDGPDYSSPLTGSG), and provide high capacity (> 5·10¹⁰ vp/mL of resin) and yield under mild elution conditions (~50% at pH 8.0). Peptide‐functionalized adsorbents prepared using different matrices (polymethylmethacrylate vs. agarose) were initially tested to compare the purification performance. AEFFIWNA‐SulfoLink resin was selected for its yield of cell‐transducing AdVs (~50%) and removal of HCPs and hcDNA (144‐fold and 56‐fold). Similarly, TNDGPDYSSPLTGSG‐Toyopearl resin afforded ~50% yield and > 50‐fold reduction of impurities. Additional gains in product purity were achieved by optimizing the washing step, which removed free hexon proteins and additional HCPs. All peptide‐functionalized resins maintained their purification performance for 10 cycles upon regeneration at pH ~2.0. The purification process was assembled to include clarification, affinity capture in bind‐and‐elute mode using AEFFIWNA‐SulfoLink resin, and polishing in flow‐through mode using mixed‐mode resins. The optimized process provided a yield ~50% of cell‐infecting units (IFU) and a product titer ~10⁷ IFU/mL, along with residual HCP and hcDNA levels (8.76 ng/mL and 44 ng per dose, respectively) that meet clinical requirements.


Systems Metabolic Engineering of Clostridium tyrobutyricum for 1,3‐Propanediol Production From Crude Glycerol

Clostridium tyrobutyricum has emerged as a non‐pathogenic microbial cell factory capable of anaerobic production of various value‐added products, such as butyrate, butanol, and butyl butyrate. This study reports the first systematic engineering of C. tyrobutyricum for the heterologous production of 1,3‐propanediol (1,3‐PDO) from industrial by‐product crude glycerol. Initially, the glycerol reductive pathway for 1,3‐PDO production was constructed, and the unique glycerol oxidation pathway in C. tyrobutyricum was elucidated. Subsequently, the glycerol metabolism and 1,3‐PDO synthesis pathways were enhanced. Furthermore, the intracellular reducing power supply and the fermentation process were optimized to improve 1,3‐PDO production. Consequently, 54.06 g/L 1,3‐PDO with a yield of 0.64 mol/mol and a productivity of 1.13 g/L·h was obtained using crude glycerol and fish meal. The strategies described herein could facilitate the engineering of C. tyrobutyricum as a robust host for synthesizing valuable chemicals.


Heterologous Expression of Candida antarctica Lipase B in Aspergillus niger Using CRISPR/Cas9‐mediated Multi‐Gene Editing

Aspergillus niger, a filamentous fungus, is known as a cell factory due to its ability to produce large amounts of organic acids and industrial enzymes. Lipase B from Candida antarctica (CALB) is one of the most widely used lipases in industrial applications, including oil processing, papermaking, food, pharmaceuticals, and personal care products. In this study, the CRISPR/Cas9 technique was employed to knock out the pyrG and kusA genes in A. niger. The CALB gene was integrated into the high‐production protein gene loci, such as glaA and amyA, to construct a multi‐copy CALB production engineered strain. Additionally, the pepA, aglU, and bglA genes were deleted, which minimized the background level of secreted proteins in A. niger and increased the production of CALB. After two rounds of gene editing, the A. niger with multi‐copy CALB was created, and the engineered A. niger CCTCC 206047.09 with high CALB yield was isolated. After 120 h of liquid fermentation, the lipase activity reached 17.84 U/mL and the protein yield reached 10.21 mg/mL. In summary, an engineered A. niger strain with high lipase activity was successfully isolated by employing a CRISPR/Cas9 system to integrate CALB into high‐expression loci, while simultaneously knocking out the host's highly expressed protein genes. These results provide an effective strategy for the high expression of both heterologous and homologous enzymes in A. niger.


Temporal Galactose‐Manganese Feeding in Fed‐Batch and Perfusion Bioreactors Modulates UDP‐Galactose Pools for Enhanced mAb Glycosylation Homogeneity

April 2025

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9 Reads

Monoclonal antibodies (mAbs) represent a majority of biotherapeutics in the market today. These glycoproteins undergo posttranslational modifications, such as N‐linked glycosylation, that influence the structural & functional characteristics of the antibody. Glycosylation is a heterogenous posttranslational modification that may influence therapeutic glycoprotein stability and clinical efficacy, which is why it is often considered a critical quality attribute (CQA) of the mAb product. While much is known about the glycosylation pathways of Chinese Hamster Ovary (CHO) cells and how cell culture chemical modifiers may influence the N‐glycosylation profile of the final product, this knowledge is often based on the final cumulative glycan profile at the end of the batch process. Building a temporal understanding of N‐glycosylation and how mAb glycoform composition responds to real‐time changes in the biomanufacturing process will help build integrated process models that may allow for glycosylation control to produce a more homogenous product. Here, we look at the effect of specific nutrient feed media additives (e.g., galactose, manganese) and feeding times on the N‐glycosylation pathway to modulate N‐glycosylation of a Herceptin biosimilar mAb (i.e., Trastuzumab). We deploy the N‐GLYcanyzer process analytical technology (PAT) to monitor glycoforms in near real‐time for bench‐scale bioprocesses operated in both fed‐batch and perfusion modes to build an understanding of how temporal changes in mAb N‐glycosylation are dependent on specific media additives. We find that Trastuzumab terminal galactosylation is sensitive to media feeding times and intracellular nucleotide sugar pools. Temporal analysis reveals an increased desirable production of single and double galactose‐occupied glycoforms over time under glucose‐starved fed‐batch cultures. Comparable galactosylation profiles were also observed between fed‐batch (nutrient‐limited) and perfusion (non‐nutrient‐limited) bioprocess conditions. In summary, our results demonstrate the utility of real‐time monitoring of mAb glycoforms and feeding critical cell culture nutrients under fed‐batch and perfusion bioprocessing conditions to produce higher‐quality biologics.


Altering 15‐Lipoxygenases to 18‐Lipoxygenases and Their Application to the Production of 5,18‐Dihydroxyeicosapentaenoic Acids

April 2025

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4 Reads

Resolvin E2 (RvE2), 5S,18R‐dihydroxyeicosapentaenoic acid (5S,18R‐DiHEPE), and 18S‐RvE2 (5S,18S‐DiHEPE) are specialized pro‐resolving mediators that function in the resolution of inflammation. These SPMs have been produced in trace amounts from eicosapentaenoic acid (EPA) using acetylated cyclooxygenase‐2 or cytochrome P450 and 5‐lipoxygenase (5‐LOX) via 18R‐ and 18S‐hydroxyeicosapentaenoic acid (18R‐ and 18S‐HEPE) intermediates. In this study, we engineered 15R‐LOX from Sorangium cellulosum and 15S‐LOX from Archangium violaceum into 18R‐LOX (L423W/L424M/L568M variant of 15R‐LOX) and 18S‐LOX (L429W/L430M/L575M variant of 15S‐LOX), respectively, via structure‐guided enzyme engineering. The engineered 18R‐LOX converted EPA into 72.5% 18R‐HEPE and 27.5% 15R‐HEPE, while the engineered 18S‐LOX formed 81.8% 18S‐HEPE and 18.2% 15S‐HEPE. Escherichia coli expressing the engineered 18R‐ or 18S‐LOX converted 4.0 or 3.0 mM EPA into 2.0 mM (641 mg/L) 18R‐HEPE or 1.8 mM (577 mg/L) 18S‐HEPE in 20 min, respectively, achieving concentrations that were > 10⁵‐fold higher than those reported previously. Furthermore, 5S‐LOX from Danio rerio (zebrafish) converted a concentration of 0.5 mM of the prepared 18R‐ or 18S‐HEPE into 0.24 mM (81 mg/L) RvE2 or 0.22 mM (74 mg/L) 18S‐RvE2 in 30 min, respectively. To the best of our knowledge, this represents the first identification of 18‐LOXs and first qualitative production of RvE2 and 18S‐RvE2.


Systematic Investigation of Impact of Antifoam and Extracellular Vesicles on Fouling of Hollow Fiber Filters in Intensified Perfusion Processes Highlights the Key Impact of Antifoam

April 2025

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13 Reads

Ultra‐high cell density perfusion cultures are becoming increasingly attractive for biologics manufacturing due to their ability to boost productivity and offer a flexible manufacturing footprint. However, these intensified perfusion processes pose challenges, particularly concerning the performance of hollow fiber filters used as cell retention devices. To facilitate their implementation in biologics manufacturing, it is crucial to understand the factors driving filter fouling and develop innovative strategies to mitigate this issue. In this study, we developed a small‐scale model to investigate various factors in cell culture that affect filter fouling. We systematically examined individual components, such as antifoam and extracellular vesicles, to assess their impact on filter performance. Our data suggest that the extent and mechanism of fouling differ among these components, likely due to variations in particle size distribution and properties. Additionally, our results indicate that simethicone‐based antifoam accumulates over time in perfusion cultures, significantly impacting fouling. We observed better cell health in perfusion runs with minimal antifoam addition. Pellet fractions isolated by ultracentrifugation at 10,000g and 100,000g from “No antifoam” perfusion culture exhibited markedly improved filter performance in the offline model, highlighting the negative impact of antifoam in perfusion cultures. Conversely, an alternative antifoam variant that does not rely on simethicone showed better filter performance in the offline model, emphasizing the role of both antifoam and membrane composition in fouling tendencies. This study is the first to systematically examine the impact of individual components in perfusion cultures on filter fouling. Further investigations will be essential to develop the next generation of robust perfusion processes.


Modeling the Migration and Growth of Shewanella Oneidensis MR‐1 in a Diffusion‐Dominated Microfluidic Gradient Chamber Under the Influence of an Antibiotic Concentration Gradient

Motility and chemotaxis allow bacteria to migrate from areas that become depleted in energy yielding substrates to more favorable locations, possibly enhancing the biodegradation of pollutants in soil and groundwater. However, in some cases substrates are co‐mingled with one or more toxic solutes that inhibit pollutant degradation and/or microbial growth, and the impacts on motility and chemotaxis represent a knowledge gap. In this study, a one‐dimensional diffusion reaction model is developed and used to simulate dissimilatory biological reduction of nitrate to ammonia (DNRA) presented in a previously published microfluidic gradient chamber (MGC) experiment, where spatial abundances of Shewanella oneidensis MR‐1 cells were recorded over 5 days in a diffusion limited porous media domain as it degraded nitrate and lactate introduced from opposite boundaries, and at one boundary co‐mixed with the antibiotic ciprofloxacin. The model considers S. oneidensis chemotaxis toward nitrate and nitrite, random motility, and growth inhibition by ciprofloxacin. Parameters were adjusted within ranges commonly reported in the literature to obtain results that agreed with the data. Simulation results indicate that motility and not chemotaxis, in combination with inhibition of cell growth by ciprofloxacin, controls the distribution of cells in the toxic region (containing ciprofloxacin) of the MGC. This suggests that cell motility may facilitate nitrate removal in soil and groundwater by enabling microorganisms to migrate toward nitrate contaminated regions with elevated antibiotic concentrations.


Schematic of a (A) blank catheter hub, (B) non‐polarized e‐catheter hub, (C) polarized e‐catheter hub operated by a micropotentiostat, and (D) HOCl microelectrode setup.
Linear sweep voltammetry and surface HOCl generation of e‐catheter hub, with WEs composed of (A) Ti, (B) Pt and (C) Au scanned at a rate of 10 mV/s.
Chronoamperometry scan of e‐catheter hubs with Au, Pt, and Ti electrodes with WEs polarized at 1.5 VAg/AgCl over 24 h for HOCl generation.
HOCl generation within e‐catheter hubs intermittently over 5 days using a commercial potentiostat with Ti, Pt, and Au electrodes.
Biocidal efficacy of HOCl generated electrochemically by e‐catheter hubs (n = 4) against A. baumannii ATCC‐17978, operated using a commercial potentiostat and equipped with (A) Ti, (B) Pt, and (C) Au electrodes. (D) Polarized e‐catheter hubs utilizing Ti, Pt, or Au wires operated by MPs.
Electrochemical Catheter Hub Operated by a Wearable Micropotentiostat Prevents Acinetobacter baumannii Infection In Vitro

April 2025

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19 Reads

Intraluminal infection of central venous catheters, used for long‐term treatment, can result in central line‐associated bloodstream infection (CLABSI). These infections can be challenging to prevent and treat due to formation of biofilms within catheter lumens, which shield bacteria from the human immune response and conventional antimicrobial therapies. Preventing bacterial colonization of catheter hubs is a strategy to prevent CLABSI. To address this, we developed a nonantibiotic, animal‐ready electrochemical catheter hub (e‐catheter hub), operated by a wearable, battery‐powered micropotentiostat (MP), that internally generates tunable hypochlorous acid (HOCl) for preventing intraluminal infection. The design evaluated three different electrode materials—titanium, platinum, and gold—for HOCl generation and biocidal activity, using working and counter electrodes of the same materials and a silver/silver chloride‐plated wire as a quasi‐reference electrode. e‐catheter hubs operated by MPs at 1.5 VAg/AgCl for 3 h generated HOCl, reducing Acinetobacter baumannii ATCC‐17978 below the detection limit (average reduction of 4.40 ± 0.05 log10 CFU/mL). The efficacy of e‐catheter hubs operated by MPs in generating HOCl and achieving biocidal activity is comparable to that of a commercial potentiostat. This study represents the first step in developing a localized, nonantibiotic strategy to mitigate CLABSI risk.


Efficient One‐Step Production of 7S,17S‐ and 10S,17S‐Dihydroxydocosahexaenoic Acids by a Double‐Oxygenating 15S‐Lipoxygenase From Chlamydomonas incerta

April 2025

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7 Reads

Specialized pro‐resolving mediators (SPMs), such as resolvin D5 (7S,17S‐dihydroxydocosahexaenoic acid, 7S,17S‐DiHDHA; RvD5) and protectin DX (10S,17S‐DiHDHA; PDX), are critical in resolving inflammation in humans. In this study, a unique double‐oxygenating 15S‐lipoxygenase (15S‐LOX) from the alga Chlamydomonas incerta was identified and characterized for its ability to simultaneously produce RvD5 and PDX from docosahexaenoic acid (DHA). Recombinant Escherichia coli expressing the C. incerta 15S‐LOX demonstrated enhanced RvD5 and PDX production under the following optimized reaction conditions: pH 8.0, 25°C, 0.5 g dry cells/L, 7.0 mM DHA, 2.0% (w/v) PVP, 2.0% (v/v) DMSO, and 200 mM cysteine used as a reductant. This one‐step biocatalytic process produced 2.91 mM (1.05 g/L) RvD5 and 2.18 mM (0.78 g/L) PDX in 90 min, with a total of 5.09 mM (1.83 g/L) and a total conversion yield of 79.6% (w/w). Compared to previously reported two‐step biocatalytic processes, this one‐step process significantly enhanced the production of particular PDX with improved productivity and simplicity. Structural analysis identified residues Phe667, Ile705, and Leu713 as regioselectivity modulators for the second oxygenation step. This study demonstrates the efficiency and industrial potential of the double‐oxygenating LOX as a biocatalyst for simultaneously producing RvD5 and PDX.


Design of the aerosol‐based PBR (abPBR) for the cultivation of cyanobacteria on Luffa. (A) 3D rendering of the abPBR. (B) Exploded drawing of the components of the abPBR including external dimensions of the cultivation chamber. (C) Picture of the abPBR filled with Luffa. (D) Luffa covered with biofilm of Desmonostoc muscorum after cultivation in the abPBR.
Light intensity as a function of the distance to the light source. The light intensity was measured through three materials: Air (rectangle), Luffa (circle), and aerosol (triangle).
Temperature and humidity curve in the abPBR over time. The aerosol supply (1 L min⁻¹) was switched off after 12 h and switched on again after 24 h. The lighting was provided from outside (see Section 2.1) and was switched off for the first 12 h and then switched on again for 12 h.
Experimental and simulative determination of the residence time distribution in abPBR. (A and C) Experimentally determined RTD and CDF in the upper third of the abPBR (abPBRtop, A) or in the lower third of the abPBR (abPBRbottom, C), calculated RTD and CDF for an ideal stirred reactor (idSTR) using the volume of the abPBR, calculated CDF for an ideal STR considering the dead space volume (real STR) according to Müller‐Erlwein (2015) and simulated RTD and CDF for abPBR (Simulation). (B) µCT Scan of a piece of Luffa (1 × 1 cm side length) with filled inner pores (red) used for the calculation of physical parameters for the simulation of RTD and CDF. (D) Simulated flow lines in the abPBR with compressed air flow at 1 L min⁻¹.
Area time yield based on the cultivation surface for the cultivation of Nostoc sp. in different biofilm‐based photobioreactors. Media supply via a submerged cultivation (stirred tank reactor, lab‐scale moving bed PBR (lsMBPBR) and pilot‐scale moving bed PBR (psMBPBR) (Walther et al. 2022) or via an aerosol produced with an ultrasonic transducer (emersed PBR [ePBR] with different cultivation surfaces [Strieth et al. 2021] and aerosol‐based PBR with Luffa [abPBR]). n = 3.
A New Aerosol‐Based Photobioreactor for the Cultivation of Cyanobacteria on Luffa

April 2025

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3 Reads

Cyanobacteria are promising organisms for sustainable biotechnology due to their ability to grow photoautotrophically and their wide range of products. Many cyanobacteria grow in the form of biofilms, which is why the development of photobioreactors (PBR) for the cultivation of cyanobacteria in the form of biofilms is of great interest. However, these biofilm PBR are mostly based on artificial growth surfaces, whereas biodegradable growth surfaces would be favored in terms of sustainable production and application. Luffa sponges (the dried fruit of Luffa cylindrica) are excellent biodegradable growth surfaces for cyanobacteria. Therefore, a biofilm PBR for cultivation of cyanobacteria on Luffa was developed in this study. Since many cyanobacteria grow naturally as biofilms in an air‐exposed form and this should be imitated to improve growth, an aerosol‐based PBR (abPBR) should be used for cultivation. This involves supplying the cyanobacteria with a nutrient mist. The abPBR was comprehensively characterized by determining the distribution of light, humidity and temperature inside the reactor. In addition, the residence time distribution of the aerosol was determined both experimentally and simulatively. In final cultivation experiments, it was shown that the abPBR is ideal for cultivating cyanobacteria and at the same time the aerosol system enables a simple imitation of drought stress. With the cyanobacteria Nostoc spec. and Desmonostoc muscorum, maximum area‐time‐yields (ATY) in relation to the growth surface of 6.34 and 4.19 g m⁻² d⁻¹, respectively, were achieved. Compared to previously developed abPBR, the ATY has been increased by a factor of 2.3.


Generating Multispecific Antibodies Through Column‐Based Redox Reactions: Part I

April 2025

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5 Reads

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1 Citation

Multispecific antibodies are increasingly being explored in the pharmaceutical industry for unmet patient needs. This study focuses on generating these molecules through an electrostatic‐steering strategy, where two separate parent homodimer antibodies are expressed and purified, then combined into the heterodimer multispecific through reduction and oxidation chemistry. Traditional manufacturing operations for electrostatic steering multispecifics can include complex processing steps. Therefore, a novel redox process to generate the multispecific has been explored. This process involves a column‐based reduction reaction and a spike of oxidant in the elution pool to form the heterodimer. This new strategy can simplify the downstream purification process for electrostatic‐steering based molecules. The method consists of simultaneously binding two separate parental homodimers to the protein A chromatography resin and applying a reductant wash to reduce the interchain disulfide bonds. The molecules are then eluted, neutralized, and oxidized to form the intact heterodimer. The mechanism and rates of reduction, heterodimerization, and oxidation have been characterized to maximize conversion and product quality. This strategy has been demonstrated successfully for five multispecifics with diverse specificity and IgG subclasses. Implementing this method for pharmaceutical bioprocesses in the production of multispecific molecules offers the potential for the reduction in manufacturing complexity while maintaining acceptable product quality and yield.


Improving Multispecific Antibody Bioprocesses Through Coculture and Column‐Based Redox Reactions: Part II

April 2025

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17 Reads

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1 Citation

Multispecifics are increasingly being evaluated in the pharmaceutical industry due to their unique mechanisms of action, enabled by their multiple antigen‐binding capabilities. The complexity of these molecules can make production challenging, prompting the development of various generation approaches. This study employs an electrostatic‐steering generation method, where charge‐based differences between two parental homodimer antibodies drive correct heterodimerization during a redox reaction of the partially purified parental homodimers. This strategy can achieve high conversion to the heterodimer with minimal product‐related impurities. However, it also necessitates separate bioreactors for each parental homodimer, leading to complex manufacturing campaigns. This work introduces a novel bioprocess for electrostatic‐steering‐based multispecifics, combining two unique components. First, two separate cell lines are cocultured, leading to the simultaneous production of both parental homodimers in a single bioreactor. The second component involves a column‐based redox reaction, where the homodimers are captured, and their disulfide bonds are reduced while bound to the protein A resin using a reductant wash. The column is then eluted and neutralized, allowing the reduced parental homodimers to heterodimerize. Finally, the addition of an oxidant enables the reformation of disulfide bonds, completing the formation of the multispecific. This new process is robust and efficient across both the lab bench and manufacturing scales, maintaining well‐controlled impurity profiles. Homodimer harvest ratios were consistently within 10%–15% of the target across various cocultured cell lines. Conversions from homodimers to heterodimers exceeded 90%, and multispecific percentages in all tested drug substance pools were above 95%. This strategy aligns the new multispecific bioprocess with typical antibody‐like processes, optimizing clinical and commercial manufacturing resources while producing complex multispecific molecules with minimal impurities.


Experimental overview of the setup. Each dMSSC chip (top right panel) is composed of six microfluidic structures (Blöbaum et al. 2024). Each oscillation structure (on the right) is composed of 6 arrays of 23 chambers for cell growth. Strains CEN.PK113‐7D, Ethanol Red, and PE2 were grown in substrate and pH dynamic environments with oscillations ranging between 0.75 and 48 min. Each strain carried a biosensor for ATP levels, glycolytic flux, or oxidative stress response. The mechanism of action of biosensors is detailed in Figure 3 and in Supporting Information Text (Additional File S1).
Selection of experimental conditions. (A) Number of doublings by CEN.PK113‐7D grown at different glucose concentrations (n = 4–8 cells across three replicates). Doublings considered the initial cells inoculated in each chamber only. (B) Percentage of alive and dead CEN.PK113‐7D cells after 1, 8, and 16 h in MSCC static cultivation at various pH. Values are based on triplicates (three individual chambers) and correspond to the mean ± standard deviation.
Growth and morphology response in dynamic environments. (A) Violin plots highlight the performance of each function (budding ratio, area, and circularity) for each cell of a specific strain under all tested dynamic environments. Positive control data (static environment) are not included but are found in Figure S5. Red dots denote the mean performance across cells in all replicates and frequencies. Student's t‐test was performed to assess differences between each pair of strains; **p ≤ 0.01, ****p ≤ 0.0001. (B) Detailed overview of the budding ratio in strains exposed to substrate and pH dynamic environments and positive control (pH 5 for pH oscillations, 50 g/L glucose for substrate oscillations). Standard deviation refers to data distribution across five replicates.
Biosensor mode of action and response in dynamic environments. (A) Overview and mechanisms of action of biosensors for ATP (QUEEN‐2m, a circularly‐permuted fluorescent protein), glycolytic flux (GlyRNA, a fructose‐bis‐phosphate (FBP)‐sensitive aptameric biosensor), and oxidative stress (OxPro, an oxidative stress‐sensitive synthetic‐promoter‐based biosensor). (B) Relative ATP, glycolytic flux, and oxidative stress of the three selected strains upon substrate (left) and pH (right) dynamics. Violin plots highlight single‐cell performance. Positive control data (static environment) are not included (see Figure S6). Red dots denote the mean performance across cells in all replicates and dynamic oscillation frequencies. Student's t‐test was performed to assess differences between each pair of strains; ****p ≤ 0.0001. (C and D) Relative glycolytic flux in Ethanol Red (C) and relative oxidative stress response in CEN.PK113‐7D (D) exposed to substrate and pH dynamic environments and a static positive control (pH 5 for pH oscillations, 50 g/L glucose for substrate oscillations). Standard deviation refers to data distribution across five replicates.
Intracellular parameter performance and robustness relationship. (A) Visual representation of robustness types. For a desired function (e.g., ATP levels) and set of conditions (e.g., different oscillation frequencies), it is possible to use the robustness quantification method to measure the stability of a function across conditions R(c), populations R(p), and over time R(t). (B–D) Correlation between performance (x‐axis) and robustness types (y‐axis) for relative ATP levels (B), glycolytic flux (C), and oxidative stress response (D). For each intracellular parameter, robustness across conditions, R(c) (top), over time, R(t) (middle), and across populations, R(p) (bottom), are shown. Standard deviation represents the distribution across five oscillation frequencies only (no positive static control included) with five replicates each.
Physiology and Robustness of Yeasts Exposed to Dynamic pH and Glucose Environments

April 2025

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27 Reads

Gradients negatively affect performance in large‐scale bioreactors; however, they are difficult to predict at laboratory scale. Dynamic microfluidics single‐cell cultivation (dMSCC) has emerged as an important tool for investigating cell behavior in rapidly changing environments. In the present study, dMSCC, biosensors of intracellular parameters, and robustness quantification were employed to investigate the physiological response of three Saccharomyces cerevisiae strains to substrate and pH changes every 0.75–48 min. All strains showed higher sensitivity to substrate than pH oscillations. Strain‐specific intracellular responses included higher relative glycolytic flux and oxidative stress response for strains PE2 and CEN.PK113‐7D, respectively. Instead, the Ethanol Red strain displayed the least heterogeneous populations and the highest robustness for multiple functions when exposed to substrate oscillations. This result could arise from a positive trade‐off between ATP levels and ATP stability over time. The present study demonstrates the importance of coupling physiological responses to dynamic environments with simultaneous characterization of strains, conditions, individual regimes, and robustness analysis. All these tools are a suitable add‐on to traditional evaluation and screening workflows at both laboratory and industrial scale, and can help bridge the gap between these two.


Flux Sampling Suggests Metabolic Signatures of High Antibody‐Producing CHO Cells

April 2025

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18 Reads

Chinese hamster ovary (CHO) cells remain the industry standard for producing numerous therapeutic proteins, particularly monoclonal antibodies (mAbs). However, achieving higher recombinant protein titers remains an ongoing challenge and a fundamental understanding of the cellular mechanism driving improved bioprocess performance remains elusive. To directly address these challenges and achieve substantial improvements, a more in‐depth understanding of cellular function within a bioprocess environment may be required. Over the past decade, significant advancements have been made in the building of genome‐scale metabolic models (GEMs) for CHO cells, bridging the gap between high information content 'omics data and the ability to perform in silico phenotypic predictions. Here, time‐course transcriptomics has been employed to constrain culture phase‐specific GEMs, representing the early exponential, late exponential, and stationary/death phases of CHO cell fed‐batch bioreactor culture. Temporal bioprocess data, including metabolite uptake and secretion rates, as well as growth and productivity, has been used to validate flux sampling results. Additionally, high mAb‐producing solutions have been identified and the metabolic signatures associated with improved mAb production have been hypothesized. Finally, constraint‐based modeling has been utilized to infer specific amino acids, cysteine, histidine, leucine, isoleucine, asparagine, and serine, which could drive increased mAb production and guide optimal media and feed formulations.


Directed Evolution of an (R)‐Selective Transaminase Toward Higher Efficiency of Sitagliptin Analog Biosynthesis

Transaminase (TA)‐catalyzed asymmetric amination is considered as a green chemistry approach to synthesize pharmaceutical analogs, but their ability to accept substrate for catalyzing sterically hindered ketones remains a challenge. Sitagliptin is an antihyperglycemic drug to treat type II diabetes. Herein, we exploited an efficient (R)‐selective TA to biosynthesize sitagliptin analog (R)‐3‐amino‐1‐morpholino‐4‐(2,4,5‐trifluorophenyl)butan‐1‐one. Starting from a previously constructed (R)‐ATA5, two rounds of directed evolution were performed through combining error‐prone PCR, site‐directed saturation and combinatorial mutagenesis. The resultant variant ATA5/F189H/S236T/M121H showed a 10.2‐fold higher activity and a 4‐fold improved half‐life at 45°C. Crucially, the variant was able to either catalyze the amination of 700 mM substrate with a conversion up to 93.1% and product e.e.> 99% in a cosolvent reaction system, or biotransform 200 mM substrate with a conversion of 97.6% and product e.e.> 99% in a cosolvent‐free system. Furthermore, the structural analysis gave insight into how the mutations affected enzymatic activity and thermostability. This study, which consists of constructing a robust (R)‐selective TA and the new synthesis route with the highest conversion ever reported, provides a reference for industrial manufacturing sitagliptin analog.


Design of the device and computational simulations (a) Pressure distribution in the apical chamber. (b) Shear stress distribution on the membrane wall. (c) Shear stress along with width of the membrane showing almost equal distribution. (d) Experimental setup showing recirculating flow in basolateral chamber and open loop flow in apical chamber (P‐peristaltic pump). (e) The experiment timeline shows a total of 12 days with 5 days of coculture with bacterial biofilm. (Created in BioRender. Marques, C. [2025] https://BioRender.com/a20z156J).
SIOC characterization. Images taken in the same location in the device for viability analysis (Day 10 after cell seeding) depicting (a) cell nucleus (Hoechst), (b) Calcein AM, showing live cell population (c) PI, showing dead cell population. (d) Merged image. Scale bar = 100 µm. (e) Tight junction formation (Day 7 after cell seeding) shown by anti‐occludin staining, red‐anti‐occludin, blue‐Hoechst, scale bar = 20 µm. (f) Lucifer yellow permeability comparison in the presence of L. rhamnosus in the static system, and SIOC (Day 0–5 of coculture with bacteria). (g) Epithelial differentiation shown by polarized epithelium with villi‐like structures along with L. rhamnosus at Day 2 of coculture with the bacteria (blue: mammalian cell DNA (Hoechst), green: F‐actin (Phalloidin), red: mCherry L. rhamnosus). (h) barrier differentiation shown by anti‐villin staining at Day 0 of coculture (red: villin, blue: Nucleus (Hoechst) scale bar 20 µm (i) 3D image showing barrier with thick L. rhamnosus biofilm and mucus (day 2 coculture). (blue: mammalian cell DNA (Hoechst), green: mucus (WGA), red: mCherry L. rhamnosus). Scale bar = 20 µm. Results are presented as mean ± SD of triplicate experiments. Significance was determined with two‐way ANOVA followed by Bonferroni posttest (*p < 0.05, **p < 0.01, ***p < 0.001).
Mucus staining after static or SIOC culture. AB staining in the static system at (a) Day 0, (b) Day 2, (c) Day 5 of coculture. PAS staining in the static system at (d) Day 0, (e) Day 2, (f) Day 5 of coculture. AB staining in the SIOC at (g) Day 0, (h) Day 2, (i) Day 5 of coculture. PAS staining in the SIOC at (j) Day 0, (k) Day 2, (l) Day 5 of coculture. (m) AB and (n) PAS staining of 5 days culture of only the biofilm in the microfluidic device. Mean digital intensity quantification using ImageJ for (o) AB and (p) PAS. Scale bar = 100 µm. Representative images are presented. Results are presented as mean ± SD of triplicate experiments (at least 10 images from each sample). Significance was determined with one‐way ANOVA followed by Tukey's posttest (*p < 0.05, **p < 0.01, ***p < 0.001).
Confocal image analysis of mucus. Samples of the in vitro epithelium cocultured with L. rhamnosus were stained for mucus (WGA) and MUC2 and thickness was analyzed in ImageJ. (a–c) Static samples at Day 0, Day 2 and Day 5 of coculture respectively. (d–f) SIOC samples at Day 0, Day 2 and Day 5 of coculture respectively. (g) Mucus thickness analysis from WGA stain (μm). (h) MUC2 thickness analysis from MUC2 antibody stain (μm). Blue: nuclear material, Red: MUC2, Green: Mucus layer (WGA). Scale bar = 20 µm. Representative images are presented. Results are presented as mean±SD of triplicate experiments (68 images). Significance was determined with one‐way ANOVA followed by Tukey's posttest (*p < 0.05, **p < 0.01, ***p < 0.001).
Analysis of biofilm formation on mammalian cells. Z‐stack images were acquired at Days 2 and 5 of coculture in both static system and SIOC. (a) static Day 2, (b) static Day 5, arrows indicating mCherry L. rhamnosus biofilm, (c) dynamic Day 2, (d) dynamic Day 5. At each time point various parameters of the biofilm were analyzed: (e) biomass (μm³/μm²), (f) maximum thickness from substratum (μm), (g) biofilm surface area to the total biofilm volume ratio (μm²/μm³), (h) Roughness coefficient for the biofilm. (red: mCherry L. rhamnosus; green: mucus; blue: DNA). Representative images are presented. Biofilm analysis was performed on 56 images from triplicate experiments. Significance was determined with one‐way ANOVA followed by Tukey's posttest (*p < 0.05, **p < 0.01, ***p < 0.001).
Small Intestine on a Chip Demonstrates Physiologic Mucus Secretion in the Presence of Lacticaseibacillus rhamnosus Biofilm

April 2025

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21 Reads

The small intestine is an area of the digestive system difficult to access using current medical procedures, which prevents studies on the interactions between food, drugs, the small intestinal epithelium, and resident microbiota. Therefore, there is a need to develop novel microfluidic models that mimic the intestinal biological and mechanical environments. These models can be used for drug discovery and disease modeling and have the potential to reduce reliance on animal models. The goal of this study was to develop a small intestine on a chip with both enterocyte (Caco‐2) and goblet (HT29‐MTX) cells cocultured with Lacticaseibacillus rhamnosus biofilms, which is of one of several genera present in the small intestinal microbiota. L. rhamnosus was introduced following the establishment of the epithelial barrier. The shear stress within the device was kept in the lower physiological range (0.3 mPa) to enable biofilm development over the in vitro epithelium. The epithelial barrier differentiated after 5 days of dynamic culture with cell polarity and permeability similar to the human small intestine. The presence of biofilms did not alter the barrier's permeability in dynamic conditions. Under fluid flow, the complete model remained viable and functional for more than 5 days, while the static model remained functional for only 1 day. The presence of biofilm increased the secretion of acidic and neutral mucins by the epithelial barrier. Furthermore, the small intestine on a chip also showed increased MUC2 production, which is a dominant gel‐forming mucin in the small intestine. This model builds on previous publications as it establishes a stable environment that closely mimics in vivo conditions and can be used to study intestinal physiology, food‐intestinal interactions, and drug development.


Development of an HEK293 Suspension Cell Culture Medium, Transient Transfection Optimization Workflow, and Analytics for Batch rAAV Manufacturing

April 2025

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36 Reads

Recombinant adeno associated virus (rAAV) vectors have become popular delivery vehicles for in vivo gene therapies, but demand for rAAVs continues to outpace supply. Platform processes for rAAV production are being developed by many manufacturers, and transient chemical transfection of human embryonic kidney 293 (HEK293) cells is currently the most popular approach. However, the cutting edge nature of rAAV process development encourages manufacturers to keep cell culture media formulations, plasmid sequences, and other details proprietary, which creates hurdles for small companies and academic labs seeking to innovate in this space. To address this problem, we leveraged the resources of an academic‐industry consortium (Advanced Mammalian Biomanufacturing Innovation Center, AMBIC) to develop an rAAV production system based on transient transfection of suspension HEK293 cells adapted to an in‐house, chemically defined medium. We found that balancing iron and calcium levels in the medium were crucial for maintaining transfection efficiency and minimizing cell aggregation, respectively. A design of experiments approach was used to optimize the transient transfection process for batch rAAV production, and PEI:DNA ratio and cell density at transfection were the parameters with the strongest effects on vector genome (VG) titer. When the optimized transient process was transferred between two university sites, VG titers were within a twofold range. Analytical characterization showed that purified rAAV from the AMBIC process had comparable viral protein molecular weights versus vector derived from commercial processes, but differences in transducing unit (TU) titer were observed between vector preps. The developed media formulation, transient transfection process, and analytics for VG titer, capsid identity, and TU titer constitute a set of workflows that can be adopted by others to study fundamental problems that could improve product yield and quality in the nascent field of rAAV manufacturing.


A Tool for On‐Line Monitoring Microalgal Bioprocesses Based on Gas Balance Analysis

April 2025

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59 Reads

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1 Citation

This study introduces a novel method for monitoring microalgae growth and evaluating mass transfer efficiency in photobioreactors, specifically under non‐limiting and controlled growth conditions. By leveraging data on elemental composition, gas transfer rates, and oxygen production rate, the method estimates biomass growth and assesses gas‐liquid mass transfer coefficients. The approach uses indirect measurements to infer critical parameters, including biomass concentration, total inorganic carbon, and nitrogen levels. Results demonstrate accurate predictions of biomass growth and carbon dynamics, along with effective characterization of mass transfer coefficients. This method offers a robust tool for optimizing photobioreactor performance and enhancing process control.


Schematic diagram of modularized genetic memory system in vitro.
Construction and characterization of in vitro memory systems. (A) Schematic diagram illustrating the noncovalent conjugation of matrix material and DNA fragments through streptavidin‐biotin interaction. (B) Confocal microscope images showing the assembly of streptavidin‐anchored magnetic beads with linearized DNA fragments labeled with 5‐FAM (green fluorescence). Scale bar: 10 µm. (C) Schematic diagram showing the integration of the KEY plasmids into DNA fragments. attP/L: left half of attP; attP/R: right half of attP; attB/L: left half of attB; attB/R: right half of attB; Lp: left primer for plasmid; Rp: right primer for plasmid; Lf: left primer for fragment; Rf: right primer for fragment. (D) Agarose gel electrophoresis showing the expected sizes of amplified DNA fragments: Lp‐Rp (192 bp) and Rf‐Lp (434 bp), generated via PCR using specific primer pairs. M: DNA marker. (E) Schematic design showing the assembly of parS (DNA site, labeled in yellow) and ParB (protein domain, labeled in blue). (F) Confocal microscope images showing the assembly of fused protein ParB‐GFP with DNA fragments containing the parS site. Control: DNA fragment without parS site. Scale bar: 10 µm. (G) Characterization of in vitro genetic memory systems using different integrase variants and under varying reaction temperatures. dBxb1: N‐terminal catalytic residue (serine) mutated to alanine, preserving binding activity but lacking cleavage activity. Scale bar: 10 µm.
Evaluation of optimized memory systems in vitro. (A) Design and construction of DNA fragments with a single attB site in five length variations (f1: 93 bp, f2: 250 bp, f3: 500 bp, f4: 1000 bp, f5: 2000 bp). Result of agarose gel electrophoresis shows the expected DNA fragments amplified by PCR. (B) Left: Design of DNA fragments with multiple attB sites, each unit (black bracket) is 1000 bp in length. Right: Result of agarose gel electrophoresis shows the memory integration products after recombination between KEY plasmids and DNA fragments with different attB sites. Fragments (f) are DNA sequences (1000 bp); KEY plasmids (p, 2710 bp) contain attP‐Bxb1; 1: recombinant product with Bxb1 (3191 bp), 2: recombinant product with Bxb1 and phiC31 (6062 bp), 3: recombinant product with Bxb1, phiC31, and TP901‐1 (8845 bp). attB1: Bxb1‐based; attB2: phiC31‐based; attB3: TP901‐1‐based. (C) Schematic workflow illustrating the design of in vitro memory input from a single att pair (Bxb1) to multiple att pairs (from left to right: Bxb1, phiC31, TP901‐1). (D) Effect of reaction temperature on the memory input efficiency of Bxb1, (E) phiC31, and (F) TP901‐1, respectively. All reactions were conducted over 12 h. Data are presented as mean ± s.d. with three biological replicates. Error bars represent standard deviation (s.d.).
Design and characterization of scalable in vitro memory systems. (A) Schematic diagram illustrating the scalability of the in vitro memory system. DNA fragments containing diverse information allow for information enrichment across two distinct dimensions: quantity (system 1) and category (system 2). (B) Quantitative accumulation enabled by the in vitro memory system using the same repeat pair (Bxb1‐sfGFP). Signal intensity on the material's surface increases with the integration of multiple att‐Bxb1 sites. Data are presented as mean ± s.d. with three biological replicates. Error bars represent standard deviation (s.d.). (C) Categorical accumulation enabled by the in vitro memory system using orthogonal pairs (Bxb1‐sfGFP, phiC31‐mCherry, TP901‐1‐ECFP). KEY plasmids containing attP sites and fluorescent proteins were paired separately and integrated with DNA fragments attached to streptavidin‐anchored magnetic beads. Following assembly, the beads were visualized using confocal microscopy. Scale bar: 10 µm.
Construction of a memorable biocatalytic system for (S)‐1‐phenyl‐1,2‐ethanediol synthesis. (A) Schematic diagram of a two‐step enzymatic reaction pathway involves two enzymes for converting styrene to (S)‐1‐phenyl‐1,2‐ethanediol ((S)‐PED). StyA works as a subunit enzyme of styrene monooxygenase (SMO), and 1‐benzyl‐1,4‐dihydronicotinamide (BNAH) acts as the nicotinamide cofactor and sole reductant. SpEH functions as an epoxide hydrolase. (B) Effect of styrene oxide concentration on product yield between two systems. (C) Time course of (S)‐1‐phenyl‐1,2‐ethanediol production from styrene oxide within 40 min. (D) Columns: Effect of BNAH concentration on product yield; Lines: Effect of BNAH concentration on the rate of change (absolute value). Each point represents the rate of change in product yield between the previous and current measurements. A lower value indicates higher robustness of the system. (E) Time course of (S)‐1‐phenyl‐1,2‐ethanediol production from styrene in 6 h. All reactions above were conducted with three biological replicates and data are presented as mean ± s.d. Error bars represent standard deviation (s.d.). Student's t‐tests are used for statistical analysis, and p < 0.05 indicates statistical significance (*p < 0.05, **p < 0.01).
Establishing a Serine Integrase‐Based Genetic Memory System In Vitro

The increasing demand for advanced biosystems necessitates innovative approaches to store and process genetic information. DNA, as a high‐density storage medium, offers a promising solution for creating genetic memory systems that can provide state‐dependent responses to various stimuli. To date, numerous studies have reported on genetic memory systems in living organisms. However, developing modular, orthogonal, and quantifiable in vitro genetic memory systems with scalable biological components remains a significant challenge. In this study, we present an in vitro genetic memory system utilizing three orthogonal serine integrases for DNA‐based information storage and processing. By organizing the system into three standardized modules featuring two noncovalent chemical interactions (streptavidin‐biotin and parS‐ParB), we successfully designed and tested the orthogonality, scalability, and functionalization of these systems. Notably, we expanded the application to implement a cascade biotransformation process converting styrene to (S)‐1‐phenyl‐1,2‐ethanediol ((S)‐PED) with remarkable efficiency, achieving up to double the transformation rate compared to free‐floating purified enzymes. We anticipate that these constructions hold significant potential for advancing artificial memory systems in vitro and will provide a reliable framework for the development of programmable biochemical functions in synthetic biology.


Journal metrics


3.5 (2023)

Journal Impact Factor™


28%

Acceptance rate


7.9 (2023)

CiteScore™


22 days

Submission to first decision


0.885 (2023)

SNIP


$5,060.00 / £3,330.00 / €4,250.00

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